From darkness to twilight: Morphological divergence between cave and surface-subterranean ecotone Niphargus species.

Subterranean and surface habitats are in stark contrast in several environmental factors. Therefore, adaptation to the subterranean environment typically impedes the (re)colonisation of surface habitats. The genus Niphargus includes amphipod crustaceans that primarily occupy subterranean habitats. All its species show typical adaptations to the subterranean environment. However, some Niphargus species occur in surface-subterranean ecotones. To understand whether (i) habitat-based phenotypic divergence is present between the cave and the ecotone species and (ii) similar phenotypes emerge independently in each ecotone, we studied morphological divergence between four cave and four ecotone Niphargus species based on 13 functional morphological traits. To account for different selection acting on the sexes, we included both males (N = 244) and females (N = 222). Nine out of 13 traits showed habitat-divergence. Traits related to feeding and crawling were shorter, while traits related to oxygenation were larger in ecotone species. Eleven out of 13 traits were sexually dimorphic. Traits related to oxygenation and crawling were larger in females, while the trait related to swimming was larger in males. We found that the extent of sexual dimorphism differs between the habitats in eight traits related to sensing, feeding, oxygenation and crawling. Additionally, we found that in certain traits related to sensing and oxygenation, habitat-related differences are only present in one sex, but not the other. We conclude that the detected differences between the cave and the ecotone species indicate divergent evolution, where similarities among the different species within habitat type indicate convergent evolution. The high degree of sexual dimorphism paired with differences in sexual dimorphism between the habitats in certain traits suggest that sexual and fecundity selections have comparable effects to environmental selection. Thus, studies of habitat-dependent adaptations investigating one sex only, or not considering sexual dimorphism, can lead to erroneous conclusions.

Sexual dimorphism in subterranean amphipod crustaceans covaries with subterranean habitat type.

Sexual dimorphism can evolve in response to sex-specific selection pressures that vary across habitats. We studied sexual differences in subterranean amphipods Niphargus living in shallow subterranean habitats (close to the surface), cave streams (intermediate), and cave lakes (deepest and most isolated). These three habitats differ because at greater depths there is lower food availability, reduced predation, and weaker seasonality. Additionally, species near the surface have a near-even adult sex ratio (ASR), whereas species from cave lakes have a female-biased ASR. We hypothesized (a) a decrease in sexual dimorphism from shallow subterranean habitats to cave lake species because of weaker sexual selection derived from changes in the ASR and (b) an increase in female body size in cave lakes because of stronger fecundity selection on account of oligotrophy, reduced predation, and weaker seasonality. We measured body size and two sexually dimorphic abdominal appendages for all 31 species and several behaviours related to male competition (activity, risk-taking, exploration) for 12 species. Species with an equal ASR that live close to the surface exhibited sexual dimorphism in all three morphological traits, but not in behaviour. The body size of females increased from the surface to cave lakes, but no such trend was observed in males. In cave lake species, males and females differed neither morphologically nor behaviourally. Our results are consistent with the possibility that sexual and fecundity selection covary across the three habitats, which indirectly and directly, respectively, shape the degree of sexual dimorphism in Niphargus species.

Sex-dependent increase of movement activity in the freshwater isopod Asellus aquaticus following adaptation to a predator-free cave habitat.

Populations experiencing negligible predation pressure are expected to evolve higher behavioral activity. However, when sexes have different expected benefits from high activity, the adaptive shift is expected to be sex-specific. Here, we compared movement activity of one cave (lack of predation) and three adjacent surface (high and diverse predation) populations of Asellus aquaticus, a freshwater isopod known for its independent colonization of several caves across Europe. We predicted 1) higher activity in cave than in surface populations, with 2) the difference being more pronounced in males as they are known for active mate searching behavior, while females are not. Activity was assessed both in the presence and absence of light. Our results supported both predictions: movement activity was higher in the cave than in the surface populations, particularly in males. Relaxed predation pressure in the cave-adapted population is most likely the main selective factor behind increased behavioral activity, but we also showed that the extent of increase is sex-specific.

Prevention of Chronic Morbidities in Extremely Premature Newborns with LISA-nCPAP Respiratory Therapy and Adjuvant Perinatal Strategies.

Less invasive surfactant administration techniques, together with nasal continuous airway pressure (LISA-nCPAP) ventilation, an emerging noninvasive ventilation (NIV) technique in neonatology, are gaining more significance, even in extremely premature newborns (ELBW), under 27 weeks of gestational age. In this review, studies on LISA-nCPAP are compiled with an emphasis on short- and long-term morbidities associated with prematurity. Several perinatal preventative and therapeutic investigations are also discussed in order to start integrated therapies as numerous organ-saving techniques in addition to lung-protective ventilations. Two thirds of immature newborns can start their lives on NIV, and one third of them never need mechanical ventilation. With adjuvant intervention, these ratios are expected to be increased, resulting in better outcomes. Optimized cardiopulmonary transition, especially physiologic cord clamping, could have an additively beneficial effect on patient outcomes gained from NIV. Organ development and angiogenesis are strictly linked not only in the immature lung and retina, but also possibly in the kidney, and optimized interventions using angiogenic growth factors could lead to better morbidity-free survival. Corticosteroids, caffeine, insulin, thyroid hormones, antioxidants, N-acetylcysteine, and, moreover, the immunomodulatory components of mother's milk are also discussed as adjuvant treatments, since immature newborns deserve more complex neonatal interventions.

Comparison of flexible nasogastric tube and semi-rigid catheter during less invasive surfactant administration.

BACKGROUND: Various flexible and semi-rigid catheter techniques have been reported for surfactant delivery during less invasive surfactant administration (LISA) in preterm infants. Data on the effect of catheter selection on procedural success rates and adverse events are limited. Our objective was to compare the rates of success and adverse events of LISA performed with nasogastric tube and semi-rigid catheter. METHODS: This was a post-hoc analysis of data from a quality improvement project. LISA was performed according to the standardized local protocol. Baseline characteristics, data on performance of LISA, degree of difficulty in laryngoscopy and vital parameters after the initiation of LISA were collected and outcomes were compared between groups. RESULTS: Fifty-six infants were included (21 with nasogastric tube, and 35 with semi-rigid catheter). Procedure success rate (defined as a single LISA attempt resulting in intratracheal administration of the planned dose of surfactant), incidence of adverse events, heart rate and oxygen saturation values and outcomes did not differ significantly between the two groups. When using a nasogastric tube for LISA, a significantly higher fraction of inspired oxygen was needed in the 3rd (0.62 vs. 0.48, P=0.024), 4th (0.61 vs. 0.37, P<0.001) and 5th minute (0.48 vs. 0.37, P=0.001) to maintain normal oxygen saturations. CONCLUSIONS: Use of the semi-rigid catheter was associated with better oxygenation during and shortly after the procedure. Our results may help neonatal units to develop local guidelines.

[Trends in the management of respiratory distress syndrome in very preterm infants transferred to the Clinical Center of the University of Debrecen]. / A respirációs distressz szindróma kezelésének trendjei a Debreceni Egyetem Klinikai Központjába szállított igen éretlen koraszülöttekben.

INTRODUCTION: Although in utero transport is recommended in the case of threatening preterm delivery, it is not always possible. Management during postnatal transport may influence neonatal outcomes. OBJECTIVE: The aim of this study was to investigate the trends in patient characteristics, respiratory management and outcomes in very preterm infants requiring postnatal transfer between 2008 and 2021. METHOD: We conducted a retrospective study. Data were collected from both written and electronic medical records. Trends were assessed using joinpoint regression analysis and summarized as annual percentage changes (APC). RESULTS: A total of 177 infants were included. The number of transfers per year showed non-significant increase over time (APC = 6.8%, p = 0.087). The proportion of time above 60 minutes for care provided by the transport team at the referral site significantly increased (APC = 7.4%, p = 0.016). Between 2008 and 2010, the use of mechanical ventilation during transports increased (APC = 36.4%, p = 0.578), then it showed a decreasing trend during the rest of the study period (APC = -7.2%, p = 0.068). The use of oxygen concentrations above 40% significantly decreased (APC = -9.5%, p = 0.043). The proportion of surfactant doses less than 150 mg/kg showed a decreasing trend (APC = -7.65%, p = 0.162), while doses above 180 mg/kg significantly increased over time (APC = 8.5%, p = 0.031). Neonatal long-term outcome indicators showed improving trends. DISCUSSION: We observed relevant trends toward non-invasive approaches and improving outcomes. CONCLUSION: Our study can facilitate the ongoing change of approach to care during postnatal transport, promote the development of relevant protocols and guidelines, which together can improve the outcome of preterm infants born outside tertiary care centers. Orv Hetil. 2023; 164(15): 571-576.

Synchronized intermittent mandatory ventilation with volume guarantee and pressure support in neonates: Detailed analysis of ventilator parameters.

OBJECTIVE: To analyse the relationship between peak inflating pressure, expired tidal volume, respiratory rate, and inspiratory time of volume-guaranteed ventilator inflations and pressure-supported spontaneous breaths during synchronized intermittent positive pressure mode with volume guarantee and pressure support (SIMV-VG-PS) in neonates. METHODS: Ventilator parameters were downloaded every second from 16 babies ventilated with SIMV-VG-PS mode using Dräger Babylog VN500 ventilators over 137 days. Transcutaneous carbon dioxide (tcCO2 ) data were also collected. Data were computationally analysed using Python. The average of each ventilator parameter was determined during each minute separately for ventilator inflations and for spontaneous breaths. These values were compared and their effect on tcCO2 levels was also analysed. RESULTS: The relationship between the peak inflating pressure of the volume guaranteed inflations (PIPVG ) and pressure-supported spontaneous breaths (PIPPS ) was highly variable. The PIPPS /PIPVG ratio differed significantly from the value (0.66) targeted by clinicians (group median: 0.80, range: 0.50-1.00). PIPPS frequently exceeded PIPVG . When PIPPS /PIPVG was >0.66, the expired tidal volume and the rate of the pressure-supported spontaneous breaths were also significantly (p < 0.0001) higher, but there was no difference in tcCO2 levels. The flow-cycled spontaneous breaths had significantly shorter inspiratory times than ventilator inflations. CONCLUSIONS: During SIMV-VG-PS it is difficult to ensure a pressure support level proportionate to the inflating pressure of ventilator inflations and to achieve the stability of tidal volumes.

Incidence, predictors of success and outcome of LISA in very preterm infants.

OBJECTIVES: The aim of this study was to examine the success rate of less invasive surfactant administration (LISA), to identify early predictive factors for the outcome of LISA, and to compare neonatal outcomes between the LISA failure group and the group of infants who were successfully treated with LISA. DESIGN: A retrospective cohort study. PATIENTS: Infants born at less than 33 weeks of gestation (n = 158) and treated with LISA for respiratory distress syndrome. RESULTS: LISA was successful in 86 cases (54.4%); 72 preterm infants (45.6%) needed additional surfactant therapy and/or mechanical ventilation in the first 72 h (LISA failure). In a multivariate logistic regression analysis, six independent predictors of LISA success were identified: core temperature at the time of admission (adjusted odds ratio (OR): 3.56), dose of poractant alfa (<200 mg/kg; adjusted OR: 0.254), elevated C-reactive protein (>10 mg/L) at 24 h of life (adjusted OR: 0.28), highest respiratory severity score (RSS) during the first hour of life or at the time of LISA (adjusted OR: 0.463), maternal age (adjusted OR: 0.923), and birth weight (adjusted OR: 1.003). The receiver operating curve created by using the identified factors indicates good predictive power with an area under the curve of 0.85. LISA failure was associated with a substantially higher risk of complications. CONCLUSION: LISA success can be predicted by variables available before the intervention. Failure of LISA is relatively frequent event in very preterm infants and is associated with adverse outcomes. Prevention of hypothermia during early stabilization and appropriate dosing of surfactant may increase LISA success rates and improve patient outcome.

Parallel morphological evolution and habitat-dependent sexual dimorphism in cave- vs. surface populations of the Asellus aquaticus (Crustacea: Isopoda: Asellidae) species complex.

Studying parallel evolution (repeated, independent evolution of similar phenotypes in similar environments) is a powerful tool to understand environment-dependent selective forces. Surface-dwelling species that repeatedly and independently colonized caves provide unique models for such studies. The primarily surface-dwelling Asellus aquaticus species complex is a good candidate to carry out such research, because it colonized several caves in Europe. By comparing 17 functional morphological traits between six cave and nine surface populations of the A. aquaticus species complex, we investigated population divergence in morphology and sexual dimorphism. We found habitat-dependent population divergence in 10 out of 17 traits, likely reflecting habitat-driven changes in selection acting on sensory systems, feeding, grooming, and antipredator mechanisms. Sexual dimorphism was present in 15 traits, explained by sexual selection acting on male traits important in male-male agonistic behavior or mate guarding and fecundity selection acting on female traits affecting offspring number and nursing. In eight traits, the degree of sexual dimorphism was habitat dependent. We conclude that cave-related morphological changes are highly trait- and function-specific and that the strength of sexual/fecundity selection strongly differs between cave and surface habitats. The considerable population variation within habitat type warrants further studies to reveal cave-specific adaptations besides the parallel patterns.

No room for males in caves: Female-biased sex ratio in subterranean amphipods of the genus Niphargus.

Sex allocation theory predicts that the proportion of daughters to sons will evolve in response to ecological conditions that determine the costs and benefits of producing each sex. All else being equal, the adult sex ratio (ASR) should also vary with ecological conditions. Many studies of subterranean species reported female-biased ASR, but no systematic study has yet been conducted. We test the hypothesis that the ASR becomes more female-biased with increased isolation from the surface. We compiled a data set of ASRs of 35 species in the subterranean amphipod Niphargus, each living in one of three distinct habitats (surface-subterranean boundary, cave streams, phreatic lakes) representing an environmental gradient of increased isolation underground. The ASR was female-biased in 27 of 35 species; the bias was statistically significant in 12 species. We found a significant difference in the ASR among habitats after correction for phylogeny. It is most weakly female-biased at the surface-subterranean boundary and most strongly female-biased in phreatic lakes. Additional modelling suggests that the ASR has evolved towards a single value for both surface-subterranean boundary and cave stream-dwelling species, and another value for 9 of 11 phreatic lake dwellers. We suggest that a history of inbreeding in subterranean populations might lower inbreeding depression such that kin selection favours mating with siblings. This could select for a female-biased offspring sex ratio due to local mate competition among brothers. The observed patterns in sex ratios in subterranean species make them a group worthy of more attention from those interested in sex allocation theory.

Is foraging innovation lost following colonization of a less variable environment? A case study in surface- vs. cave-dwelling Asellus aquaticus.

Behavioral innovation is a key process for successful colonization of new habitat types. However, it is costly due to the necessary cognitive and neural demands and typically connected to ecological generalism. Therefore, loss of behavioral innovativeness is predicted following colonization of new, simple, and invariable environments. We tested this prediction by studying foraging innovativeness in the freshwater isopod Asellus aquaticus. We sampled its populations along the route of colonizing a thermokarstic water-filled cave (simple, stable habitat with only bacterial mats as food) from surface habitats (variable environment, wide variety of food). The studied cave population separated from the surface populations at least 60,000 years ago. Animals were tested both with familiar and novel food types (cave food: bacterial mats; surface food: decaying leaves). Irrespective of food type, cave individuals were more likely to feed than surface individuals. Further, animals from all populations fed longer on leaves than on bacteria, even though leaves were novel for the cave animals. Our results support that cave A. aquaticus did not lose the ability to use the ancestral (surface) food type after adapting to a simple, stable, and highly specialized habitat.

The interplay between habitat use, morphology and locomotion in subterranean crustaceans of the genus Niphargus.

Locomotion is an important, fitness-related functional trait. Environment selects for type of locomotion and shapes the morphology of locomotion-related traits such as body size and appendages. In subterranean aquatic arthropods, these traits are subjected to multiple, at times opposing selection pressures. Darkness selects for enhanced mechano- and chemosensory systems and hence elongation of appendages. Conversely, water currents have been shown to favor short appendages. However, no study has addressed the variation in locomotion of invertebrates inhabiting cave streams and cave lakes, or questioned the relationship between species' morphology and locomotion. To fill this knowledge gap, we studied the interplay between habitat use, morphology and locomotion in amphipods of the subterranean genus Niphargus. Previous studies showed that lake and stream species differ in morphology. Namely, lake species are large, stout and long-legged, whereas stream species are small, slender and short-legged. We here compared locomotion mode and speed between three lake and five stream species. In addition, we tested whether morphology predicts locomotion. We found that the stream species lie on their body sides and move using slow crawling or tail-flipping. The species inhabiting lakes move comparably faster, and use a variety of locomotion modes. Noteworthy, one of the lake species almost exclusively moves in an upright or semi-upright position that resembles walking. Body size and relative length of appendages predict locomotion mode and speed in all species. We propose that integrating locomotion in the studies of subterranean species might improve our understanding of their morphological evolution.

Continental data on cave-dwelling spider communities across Europe (Arachnida: Araneae).

BACKGROUND: Spiders (Arachnida: Araneae) are widespread in subterranean ecosystems worldwide and represent an important component of subterranean trophic webs. Yet, global-scale diversity patterns of subterranean spiders are still mostly unknown. In the frame of the CAWEB project, a European joint network of cave arachnologists, we collected data on cave-dwelling spider communities across Europe in order to explore their continental diversity patterns. Two main datasets were compiled: one listing all subterranean spider species recorded in numerous subterranean localities across Europe and another with high resolution data about the subterranean habitat in which they were collected. From these two datasets, we further generated a third dataset with individual geo-referenced occurrence records for all these species. NEW INFORMATION: Data from 475 geo-referenced subterranean localities (caves, mines and other artificial subterranean sites, interstitial habitats) are herein made available. For each subterranean locality, information about the composition of the spider community is provided, along with local geomorphological and habitat features. Altogether, these communities account for > 300 unique taxonomic entities and 2,091 unique geo-referenced occurrence records, that are made available via the Global Biodiversity Information Facility (GBIF) (Mammola and Cardoso 2019). This dataset is unique in that it covers both a large geographic extent (from 35° south to 67° north) and contains high-resolution local data on geomorphological and habitat features. Given that this kind of high-resolution data are rarely associated with broad-scale datasets used in macroecology, this dataset has high potential for helping researchers in tackling a range of biogeographical and macroecological questions, not necessarily uniquely related to arachnology or subterranean biology.

Local- versus broad-scale environmental drivers of continental ß-diversity patterns in subterranean spider communities across Europe.

Macroecologists seek to identify drivers of community turnover (ß-diversity) through broad spatial scales. However, the influence of local habitat features in driving broad-scale ß-diversity patterns remains largely untested, owing to the objective challenges of associating local-scale variables to continental-framed datasets. We examined the relative contribution of local- versus broad-scale drivers of continental ß-diversity patterns, using a uniquely suited dataset of cave-dwelling spider communities across Europe (35-70° latitude). Generalized dissimilarity modelling showed that geographical distance, mean annual temperature and size of the karst area in which caves occurred drove most of ß-diversity, with differential contributions of each factor according to the level of subterranean specialization. Highly specialized communities were mostly influenced by geographical distance, while less specialized communities were mostly driven by mean annual temperature. Conversely, local-scale habitat features turned out to be meaningless predictors of community change, which emphasizes the idea of caves as the human accessible fraction of the extended network of fissures that more properly represents the elective habitat of the subterranean fauna. To the extent that the effect of local features turned to be inconspicuous, caves emerge as experimental model systems in which to study broad biological patterns without the confounding effect of local habitat features.

Taxonomic status and behavioural documentation of the troglobiont Lithobiusmatulici (Myriapoda, Chilopoda) from the Dinaric Alps: Are there semiaquatic centipedes in caves?

Lithobiusmatulici Verhoeff, 1899 is redescribed based on type material and newly collected specimens. Strandiolusjugoslavicus Hoffer, 1937, described from another cave in the same region in Bosnia and Hercegovina, is presented as a junior subjective synonym of L.matulici (syn. nov.). L.matulici is shown to be most closely related to Lithobiusremyi Jawlowski, 1933, type species of the subgenus Thracolithobius Matic, 1962. The completeness of the chitin-lines on the forcipular coxosternite is discussed as a promising character for interspecific differentiation within Lithobiomorpha. Documentation of hitherto unknown semiaquatic behaviour in L.matulici and other cave-dwelling centipede species from Herzegovinian-, Montenegrin- and Pyrenean caves is presented.

Roll with the fear: environment and state dependence of pill bug (Armadillidium vulgare) personalities.

Most studies on animal personality evaluate individual mean behaviour to describe individual behavioural strategy, while often neglecting behavioural variability on the within-individual level. However, within-individual behavioural plasticity (variation induced by environment) and within-individual residual variation (regulatory behavioural precision) are recognized as biologically valid components of individual behaviour, but the evolutionary ecology of these components is still less understood. Here, we tested whether behaviour of common pill bugs (Armadillidium vulgare) differs on the among- and within-individual level and whether it is affected by various individual specific state-related traits (sex, size and Wolbachia infection). To this aim, we assayed risk-taking in familiar vs. unfamiliar environments 30 times along 38 days and applied double modelling statistical technique to handle the complex hierarchical structure for both individual-specific trait means and variances. We found that there are significant among-individual differences not only in mean risk-taking behaviour but also in environment- and time-induced behavioural plasticity and residual variation. Wolbachia-infected individuals took less risk than healthy conspecifics; in addition, individuals became more risk-averse with time. Residual variation decreased with time, and individuals expressed higher residual variation in the unfamiliar environment. Further, sensitization was stronger in females and in larger individuals in general. Our results suggest that among-individual variation, behavioural plasticity and residual variation are all (i) biologically relevant components of an individual's behavioural strategy and (ii) responsive to changes in environment or labile state variables. We propose pill bugs as promising models for personality research due to the relative ease of getting repeated behavioural measurements.

Transcriptomic Insights into the Loss of Vision in Molnár János Cave's Crustaceans.

Animals that inhabit subterranean environments often undergo various distinct phenotypic modifications (referred to as "troglomorphy") as they transition to life in perpetual darkness. However, the molecular basis behind troglomorphy remains poorly understood, particularly in regards to the mechanisms involved in the reduction and/or loss of traits at the transcriptomic level. In this study, we investigate the transcriptional basis behind vision loss in populations of cave-dwelling crustaceans. We employ phylogenetic and transcriptomic methods on surface and cave-adapted populations of an emerging model species for biospeleology, the isopod Asellus aquaticus (Linnaeus, 1758), and the amphipod Niphargus hrabei S. Karaman, 1932. These two species show contrasting directionality in the surface-cave transition, which positions them as ideal study subjects. Asellus aquaticus is common in surface waters and is only occasionally found in caves, where its populations present different degrees of eye reduction and pigmentation. On the other hand, the eyeless N. hrabei has successfully colonized surface environments despite belonging to an almost exclusively cave-dwelling genus. By sequencing and assembling robust de novo transcriptomes we characterized differences in visual genes and pathways among surface and cave populations of the aforementioned species. Our results indicate that despite having reduced eyes, recent cave colonizer A. aquaticus is still capable of expressing functional visual opsins and major components of the phototransduction pathway within the cave. Niphargus hrabei, a species with an ancient cave origin, shows no clear indication of being capable of sight. However, the expression of putative functional visual opsins and other phototransduction genes was maintained, which suggests that this eyeless species might be capable of extraocular photoreception. With the present study, we aim to bring forth the Molnár János Cave system as a promising research avenue to improve our understanding of patterns of reduction and loss of vision in caves and other aphotic environments.

Mayaweckelia troglomorpha, a new subterranean amphipod species from Yucatán state, México (Amphipoda, Hadziidae).

A detailed description of a new stygobiont species of the amphipod family Hadziidae, Mayaweckelia troglomorpha Angyal, sp. n. is given, based on material collected in four cenotes of Yucatán federal state, México. Morphology was studied under light microscopy and with scanning electron microscopy. Morphological description is complemented with mitochondrial cytochrome c oxidase subunit I (COI) sequences as barcodes, with affinities to the related taxa and with notes on the species' ecology. Using COI Bayesian inference and genetic distance analyses, we show that the closest relative of the new species is M. cenoticola, forming a monophyletic group referring to the genus Mayaweckelia. Based on the available sequences, we also revealed that Mayaweckelia and Tuluweckelia are sister genera, standing close to the third Yucatán subterranean genus, Bahadzia. The data gathered on the habitat, distribution, abundance, and ecology will contribute to the conservation planning for M. troglomorpha Angyal, sp. n.

The role of isolation on contrasting phylogeographic patterns in two cave crustaceans.

BACKGROUND: The underlying mechanisms and processes that prompt the colonisation of extreme environments, such as caves, constitute major research themes of evolutionary biology and biospeleology. The special adaptations required to survive in subterranean environments (low food availability, hypoxic waters, permanent darkness), and the geographical isolation of caves, nominate cave biodiversity as ideal subjects to answer long-standing questions concerning the interplay amongst adaptation, biogeography, and evolution. The present project aims to examine the phylogeographic patterns exhibited by two sympatric species of surface and cave-dwelling peracarid crustaceans (Asellus aquaticus and Niphargus hrabei), and in doing so elucidate the possible roles of isolation and exaptation in the colonisation and successful adaptation to the cave environment. RESULTS: Specimens of both species were sampled from freshwater hypogean (cave) and epigean (surface) habitats in Hungary, and additional data from neighbouring countries were sourced from Genbank. Sequencing of mitochondrial and nuclear loci revealed, through haplotype network reconstruction (TCS) and phylogenetic inference, the genetic structure, phylogeographic patterns, and divergence-time estimates of A. aquaticus and N. hrabei surface and cave populations. Contrasting phylogeographic patterns were found between species, with A. aquaticus showing strong genetic differentiation between cave and surface populations and N. hrabei lacking any evidence of genetic structure mediated by the cave environment. Furthermore, N. hrabei populations show very low levels of genetic differentiation throughout their range, which suggests the possibility of recent expansion events over the last few thousand years. CONCLUSIONS: Isolation by cave environment, rather than distance, is likely to drive the genetic structuring observed between immediately adjacent cave and surface populations of A. aquaticus, a predominantly surface species with only moderate exaptations to subterranean life. For N. hrabei, in which populations exhibit a fully 'cave-adapted' (troglomorphic) phenotype, the lack of genetic structure suggests that subterranean environments do not pose a dispersal barrier for this surface-cave species.

Deciphering the genomic architecture of the stickleback brain with a novel multilocus gene-mapping approach.

Quantitative traits important to organismal function and fitness, such as brain size, are presumably controlled by many small-effect loci. Deciphering the genetic architecture of such traits with traditional quantitative trait locus (QTL) mapping methods is challenging. Here, we investigated the genetic architecture of brain size (and the size of five different brain parts) in nine-spined sticklebacks (Pungitius pungitius) with the aid of novel multilocus QTL-mapping approaches based on a de-biased LASSO method. Apart from having more statistical power to detect QTL and reduced rate of false positives than conventional QTL-mapping approaches, the developed methods can handle large marker panels and provide estimates of genomic heritability. Single-locus analyses of an F2 interpopulation cross with 239 individuals and 15 198, fully informative single nucleotide polymorphisms (SNPs) uncovered 79 QTL associated with variation in stickleback brain size traits. Many of these loci were in strong linkage disequilibrium (LD) with each other, and consequently, a multilocus mapping of individual SNPs, accounting for LD structure in the data, recovered only four significant QTL. However, a multilocus mapping of SNPs grouped by linkage group (LG) identified 14 LGs (1-6 depending on the trait) that influence variation in brain traits. For instance, 17.6% of the variation in relative brain size was explainable by cumulative effects of SNPs distributed over six LGs, whereas 42% of the variation was accounted for by all 21 LGs. Hence, the results suggest that variation in stickleback brain traits is influenced by many small-effect loci. Apart from suggesting moderately heritable (h2  ≈ 0.15-0.42) multifactorial genetic architecture of brain traits, the results highlight the challenges in identifying the loci contributing to variation in quantitative traits. Nevertheless, the results demonstrate that the novel QTL-mapping approach developed here has distinctive advantages over the traditional QTL-mapping methods in analyses of dense marker panels.